Variation in the Floral Morphology of Prosthechea karwinskii (Orchidaceae), a Mexican Endemic Orchid at Risk

Prosthechea karwinskii is an orchid endemic to Mexico, threatened by the destruction of its habitat and the extraction of specimens to meet its demand for ornamental and religious use. Most of its populations, including the most locally abundant ones, are found in Oaxaca state. Variations in some floral traits have been observed in these populations. We implemented a morphometric analysis to assess their floral variation and identify the most significant characters in the morphological patterns of this orchid. Floral samples were collected from 17 populations of P. karwinskii in Oaxaca, as well as from specimens used as ornaments during Easter in an Oaxacan community (Zaachila), whose origin is unknown. Sampling of natural populations covered the environmental, geographic, and morphological variation of the species. We performed an analysis of variance (ANOVA), principal component analysis (PCA), canonical variate analysis (CVA), and cluster analysis, including 185 individuals and 45 variables (12 of them were discarded in the multivariate analyses due to high correlation). Characters of the column, lateral sepal, and labellum were most informative for the observed morphological patterns. Albarradas showed the greatest morphological differentiation, mainly due to the column. In general, individuals from the same locality tended to overlap more, especially the populations of Jaltianguis and Yahuiche, which were different from the geographically close population of Etla. Teposcolula presented the highest values in perianth characters, unlike Sola_Rancho Viejo. The specimens recovered from religious ornaments were morphologically more similar to those from Yanhuitlan and Etla. This morphometric analysis identified characters as potential taxonomic markers for P. karwinskii and related species, showing its potential to associate specimens of unknown origin with their probable geographical region. Our work encourages working on collaborative conservation strategies to ensure the long-term permanence of both the species and its traditional uses.


Introduction
Prosthechea karwinskii (Mart.)J.M.H. Shaw is an orchid endemic to western and southern Mexico, inhabiting mountainous regions where it grows as a hanging epiphyte in oak or oak-pine forests subject to a well-marked seasonal drought (Figure 1A).The inflorescence is a two-flowered (exceptionally three flowers) raceme arising from the last developed pseudobulb (Figure 1B).The taxon was described in 1830, but its taxonomic history has been linked to that of Prosthechea citrina (Lex.)W.E. Higgins, or any of its nomenclatural synonyms, a very similar species, with which it becomes sympatric in some locations in Guerrero and Michoacán.In the past, the information available for P. karwinskii was invariably attributed to P. citrina; only in recent years have both species come to be considered different [1,2].This species is one of the most distinctive orchids in the Mexican flora, valued for the ornamental beauty and pleasant aroma of its flowers, as well as having cultural significance since pre-Hispanic times.It was one of the orchids from which mucilage was obtained and used as an adhesive in feather art in pre-Hispanic times and during the colonial period [3].In traditional medicine, it has been used to soothe coughs, heal wounds and burns, treat diabetes, prevent miscarriage, and aid in childbirth [4][5][6][7].The flowers are used as decorations in homes, commercial stands, and temples during Easter commemorations [8,9].Additionally, due to the beauty, color, and aroma of its flowers, this plant is cultivated in a rustic manner in orchards in many communities in Oaxaca.
Plants 2024, 13, x FOR PEER REVIEW 2 of 18 developed pseudobulb (Figure 1B).The taxon was described in 1830, but its taxonomic history has been linked to that of Prosthechea citrina (Lex.)W.E. Higgins, or any of its nomenclatural synonyms, a very similar species, with which it becomes sympatric in some locations in Guerrero and Michoacán.In the past, the information available for P. karwinskii was invariably attributed to P. citrina; only in recent years have both species come to be considered different [1,2].This species is one of the most distinctive orchids in the Mexican flora, valued for the ornamental beauty and pleasant aroma of its flowers, as well as having cultural significance since pre-Hispanic times.It was one of the orchids from which mucilage was obtained and used as an adhesive in feather art in pre-Hispanic times and during the colonial period [3].In traditional medicine, it has been used to soothe coughs, heal wounds and burns, treat diabetes, prevent miscarriage, and aid in childbirth [4][5][6][7].
The flowers are used as decorations in homes, commercial stands, and temples during Easter commemorations [8,9].Additionally, due to the beauty, color, and aroma of its flowers, this plant is cultivated in a rustic manner in orchards in many communities in Oaxaca.In most of the localities where P. karwinskii grows, it is locally scarce and survives in forest fragments surrounded by a matrix of environments modified by anthropogenic causes (conversion into crop fields, human settlements, opening of new roads, goat farming).In Oaxaca, the most abundant populations of the species occur, but they face the risk of extraction for temporary adornment in local trade [8][9][10], for religious purposes [8], and to a lesser extent, for medicinal use [4].This practice occurs annually, mainly during the species' flowering season, leading to its inclusion in the list of species of Mexican wild flora at risk [11].For other epiphytic orchids growing in the mountain forests of Mexico, it has been demonstrated that extraction for local trade has effects on species subjected to this practice, such as reductions in population size and rates of fertility and recruitment, as well as loss of genetic diversity [12][13][14][15].
In the localities of P. karwinskii in Oaxaca, variation has been observed in some floral traits, such as flower size and coloration, the shape of the labellum, and the shape of the apical teeth of the column.This leads to the assumption of the existence of intraspecific variation, at least among populations in the state, which has not been analyzed either through the use of morphological or molecular markers.The analysis of this variation will be important for identifying morphotypes with ornamental potential and desirable in a management program, recognizing phytogenetic diversity present in the species, identifying forms or subspecies within the orchid, as well as determining a possible geographical pattern associated with morphological variation.In most of the localities where P. karwinskii grows, it is locally scarce and survives in forest fragments surrounded by a matrix of environments modified by anthropogenic causes (conversion into crop fields, human settlements, opening of new roads, goat farming).In Oaxaca, the most abundant populations of the species occur, but they face the risk of extraction for temporary adornment in local trade [8][9][10], for religious purposes [8], and to a lesser extent, for medicinal use [4].This practice occurs annually, mainly during the species' flowering season, leading to its inclusion in the list of species of Mexican wild flora at risk [11].For other epiphytic orchids growing in the mountain forests of Mexico, it has been demonstrated that extraction for local trade has effects on species subjected to this practice, such as reductions in population size and rates of fertility and recruitment, as well as loss of genetic diversity [12][13][14][15].
In the localities of P. karwinskii in Oaxaca, variation has been observed in some floral traits, such as flower size and coloration, the shape of the labellum, and the shape of the apical teeth of the column.This leads to the assumption of the existence of intraspecific variation, at least among populations in the state, which has not been analyzed either through the use of morphological or molecular markers.The analysis of this variation will be important for identifying morphotypes with ornamental potential and desirable in a management program, recognizing phytogenetic diversity present in the species, identifying forms or subspecies within the orchid, as well as determining a possible geographical pattern associated with morphological variation.
Morphometric studies have been employed in some species of Orchidaceae, primarily analyzing variation associated with floral morphology [16][17][18][19][20][21][22], although vegetative mor-Plants 2024, 13,1984 3 of 17 phology has also been considered, including attributes of leaf anatomy [17,22].Studies in this regard seek to find intraspecific differences [18,23] and interspecific differentiation to delimit similar taxa considered as cryptic species [16,22], recognize taxa of hybrid origin [24], or identify morphotypes with phytogenetic value [19,20].The quantitative analysis of characters identified in the labellum has been valuable for recognizing and characterizing intraspecific variation in orchids [19,20], although the use of traits present in other floral structures has also been useful [18].Morphometry has also been used to trace the geographical origin of samples of unknown origin, particularly for species or products of economic importance [25][26][27][28][29][30].Although various sources of information (e.g., genetic and chemical) and analytical tools can address this issue, the use of multivariate methods with morphological characters offers the advantage of low cost [27][28][29][30] and relatively easy data collection for a large number of individuals [26,27].However, this topic is analytically complex and requires caution in its implementation and interpretation due to the need for a robust reference and the requirements of the analyses [26,28].
The objective of this study was to analyze the variation among populations from different localities in Oaxaca, Mexico, and to identify variables with taxonomic potential, through a morphometric analysis.Additionally, an attempt was made to determine if this set of characters allows relating specimens extracted from their habitats and whose origin is unknown, which were recovered after having had a religious use.Predictions for this study are as follows: (1) the floral morphology of P. karwinskii, analyzed using morphometric methods, will allow us to recognize the interpopulation variation of the species; and (2) the floral traits of P. karwinskii could serve as a morphological marker to associate the geographical origin of specimens from unknown localities.

ANOVA and Kruskal-Wallis Tests
Most of the 40 floral variables analyzed with ANOVA showed significant differences among the localities of P. karwinski (Table 1).However, the characters lengths between maximum width and apex (SlLa) and angle at the apex (SlAa) of the lateral sepal, width at 1/3 (SdA1), length between maximum width and apex (SdLa), and angle at the apex (SdAa) of the dorsal sepal, and width at the base of the middle lobe of the labellum (LaAbm) did not show significant differences among localities.The specimens from Teposcolula exhibited the highest values for the length and width of floral segments, which determine flower size, such as total length (SlLt) and maximum width (SlAm) of the lateral sepal, total length (SdLt) and maximum width of the dorsal sepal (SdAm), total length (PeLt) and maximum width (PeAm) of the petal, total length (LaLt) and maximum width (LaAm) of the labellum, and total length of the column (CoLt) (see Table S1).In contrast, the specimens from Sola_Rancho Viejo appeared to have the smallest flowers, as characters like total length (SdLt) and maximum width (SdAm) of the dorsal sepal, total length (PeLt) and maximum width (PeAm) of the petal, total length (LaLt), and maximum width (LaAm) of the labellum showed the lowest values (see Table S1).Additionally, the locality of Albarradas showed the highest values in two characters of the labellum (LaAml, LaA1l), in the separation between the teeth of the column (AnDlDm) and in the width of the cuniculus near the base (CuA1).However, it presented the lowest values for one character of the petal (PeAa), two in the labellum (LaAul, LaAlm) and three in the teeth of the column (DlAl, DlAn, DmAl).
The Kruskal-Wallis test revealed significant differences among localities (df = 11, p-value < 0.05) for the thickness at the middle part (CoGm, X 2 = 39.759) and the anther level of the column (CoGa, X 2 = 40.521),and width of its middle tooth (DmAn, X 2 = 27.5236).However, there were no differences among localities (df = 11, p-value > 0.05) for the apex angle of the middle lobe of the labellum (LaAam, X 2 = 18.374) and width of the column at the stigma level (CoAe, X 2 = 17.823).The specimens from Teposcolula and Sola_El Lazo recorded the highest values for the thickness of the column at the anther level (CoGa), while the individuals from Zaachila and Albarradas had the lowest values for this character (Table S1).For thickness at the middle part of the column (CoGm), the individuals from Teposcolula had the highest value.For the width of the middle tooth of the column (DmAn), the individuals from Sola_El Lazo had the highest value, while those from Albarradas had the lowest value.Both the PCA and CVA produced similar results whether the samples obtained in Zaachila were excluded or included.Therefore, for both methods, only the results of the analyses including the floral samples obtained from this community in Oaxaca are presented.For the final analyses (PCA and CVA), 185 individuals and 33 morphological variables of P. karwinskii were included.
The PCA showed that eight principal components had eigenvalues > 1.0, which together accounted for 71.91% of the total variance (Table 2).Among the eight components that retained the highest percentage of variance, the first one explained a third of it (33.51%)and was more correlated with variables related to floral size.Specifically, the maximum width of the labellum (LaAm), the petal (PeAm), and the lateral sepal (SlAm) were the most important variables in the morphological patterns observed in this axis.This analysis showed a high overlap among individuals from different populations; however, some individuals from Teposcolula and the populations of Yanhuitlan, Etla, and Sola_Rancho Viejo appeared at the extremes (Figure 2).In the CVA, three canonical axes had eigenvalues > 1.0 and together explained 66.46% of the total variance (Table 2).The first canonical axis explained 33.56% of the variance and reflected a greater contribution of the height of the middle tooth of the column (DmAl), the maximum width of both the lateral sepal (SlAm) and the lateral lobe of the labellum (LaAml).On this axis, there was a clear separation of individuals from the Albarradas population from the other populations (Figure 3A).The samples from Jaltianguis and Yahuiche showed less dispersion along the first three canonical axes and overlapped with each other.Along axis 1, individuals from these two populations were completely separated from Etla and Zaachila (except for one individual from Jaltianguis).Axis 1 also showed a complete separation of the samples from Zaachila from those from Juquila and the populations of Sola (El Lazo and Lachixío), with only a marginal overlap with the remaining Sola population (Rancho Viejo) and Tlaxiaco.Except for one individual, Teposcolula was also almost completely separated from Zaachila and Etla.maximum width of the labellum (LaAm), the petal (PeAm), and the lateral sepal (SlA were the most important variables in the morphological patterns observed in this This analysis showed a high overlap among individuals from different populations; h ever, some individuals from Teposcolula and the populations of Yanhuitlan, Etla, Sola_Rancho Viejo appeared at the extremes (Figure 2).The second axis explained 19.96% of the variance and showed a greater contribution from the total length of both the labellum (LaLt) and the lateral sepal (SlLt), as well as the height of the lateral tooth of the column (DlAl).On axis 2, individuals from the Yahuiche and Jaltianguis populations were completely separated from Tlaxiaco and Teposcolula (Figure 3A) and present a marginal overlap with Juquila and Sola_El Lazo.On this axis, Yanhuitlan is completely separate from Teposcolula and partially overlaps with Sola_Rancho Viejo.The third axis accounted for 12.94% of the variance and had as the most important variables the angle between the lateral lobe and the claw of the labellum (LaAul), the thickness of the column at the level of the anther (CoGa) and the maximum width of The second axis explained 19.96% of the variance and showed a greater contribution from the total length of both the labellum (LaLt) and the lateral sepal (SlLt), as well as the height of the lateral tooth of the column (DlAl).On axis 2, individuals from the Yahuiche and Jaltianguis populations were completely separated from Tlaxiaco and Teposcolula (Figure 3A) and present a marginal overlap with Juquila and Sola_El Lazo.On this axis, Yanhuitlan is completely separate from Teposcolula and partially overlaps with Sola_Rancho Viejo.The third axis accounted for 12.94% of the variance and had as the most important variables the angle between the lateral lobe and the claw of the labellum (LaAul), the thickness of the column at the level of the anther (CoGa) and the maximum width of the labellum (LaAm).On this axis, the separation of individuals from Albarradas from all other populations was again highlighted (Figure 3B), as seen on axis 1.Along the first three axes, the samples from Zaachila showed greater overlap with those from the populations of Yanhuitlan and Etla (Figure 3).
The cluster analysis also revealed the high morphological divergence of Albarradas population (Figure 4).In the UPGMA dendrogram, this population was externally linked to the group containing the remaining populations.This latter group is divided into three subgroups: the first formed by intermixed populations from the southern regions of Oaxaca (Sola_El Lazo and Juquila) and two populations from the Mixteca (Teposcolula and Tlaxiaco); the second includes the remaining populations from Sola de Vega (Sola_Rancho Viejo and Sola_Lachixío) nested between those of the Sierra Norte (Jaltianguis and Yahuiche); and the third subgroup comprises Zaachila linked to Yanhuitlan, with Etla also joined to them.

Morphological Variation in Natural Populations of Prosthechea karwinskii
Plants have the ability to modify their phenotype in response to environmental con ditions.However, the variation within a species due to the environment is expected to be smaller and more limited in floral characters compared to vegetative ones, since the for mer are related to reproductive success and must maintain their function [31,32].The variation in floral morphology can be interpreted as an adaptation to selection by differen pollinators [33,34].Interactions between plant-pollinators and climatic influence can ex plain the variation in floral traits, suggesting that the variation expressed on them is a product of an adaptive response [35].However, other processes can produce divergence among geographically separated populations of flowering plants, such as random genetic drift, isolation, indirect selection, and genetic factors [36,37].Because floral traits are con sidered phenotypically more stable than vegetative ones, their variation tends to be less

Morphological Variation in Natural Populations of Prosthechea karwinskii
Plants have the ability to modify their phenotype in response to environmental conditions.However, the variation within a species due to the environment is expected to be smaller and more limited in floral characters compared to vegetative ones, since the former are related to reproductive success and must maintain their function [31,32].The variation in floral morphology can be interpreted as an adaptation to selection by different pollinators [33,34].Interactions between plant-pollinators and climatic influence can explain the variation in floral traits, suggesting that the variation expressed on them is a product of an adaptive response [35].However, other processes can produce divergence among Plants 2024, 13, 1984 8 of 17 geographically separated populations of flowering plants, such as random genetic drift, isolation, indirect selection, and genetic factors [36,37].Because floral traits are considered phenotypically more stable than vegetative ones, their variation tends to be less within populations, making them valuable for recognizing infraspecific variation [19,23].Studies evaluating infraspecific morphological variation in orchids have so far been conducted using floral traits and have generally been useful in identifying such variation [16][17][18][19][20][21][22][23][24][25].
Of the floral characters evaluated in the ANOVA, most (38 out of 40) showed significant variation among the populations of P. karwinskii analyzed here, as demonstrated by the ANOVA and Kruskal-Wallis tests.Of the other five floral characters that did not show normality and were evaluated with the Kruskal-Wallis test, three of them (corresponding to the column) showed significant differences among populations (thickness in the middle part, thickness at the level of the anther, and width of the middle tooth).These tests identified Teposcolula (Mixteca region) as the population in Oaxaca that includes individuals with the largest flowers (showing the highest values for sepal, petal, and labellum length and width), while those from Sola_Rancho Viejo (Sierra Sur region) include individuals with the smallest flowers.Among orchids of ornamental value, specimens with larger flowers are preferred for cultivation and are selected as mother plants or as parents for artificial hybrids.Since P. karwinskii is an orchid appreciated for its ornamental value, the Teposcolula population holds greater importance in horticulture compared to other localities in Oaxaca.
The PCA conducted with the set of floral characters for P. karwinskii showed a high overlap between individuals from the 17 sampled locations and those obtained from Zaachila.Thus, it seems that the traits of floral morphology do not allow for detecting differences between populations, or their number is not sufficient to discriminate the infraspecific variation of P. karwinskii.Nevertheless, along the axis that accumulated the highest percentage of variance, several individuals from Teposcolula and Yanhuitlan appear at the extremes.The characters associated with this pattern were related to the width of the perianth segments (labellum, petal and lateral sepal).Among all the populations analyzed, Teposcolula has the widest segments, while Yanhuitlan, Etla, and Sola_Rancho Viejo have the narrowest segments, as also revealed by the univariate analyses.Ibáñez [27] suggested that complex patterns revealed through multivariate analyses of morphological data may be associated with the life history of the organisms.The localities of Teposcolula and Yanhuitlan host two of the largest populations of the species in Oaxaca, where the highest percentage of individuals are reproductive.Possibly, these two populations present high levels of genetic variation, which could be related to how their individuals are dispersed in the PCA graph.Other studies that have evaluated interspecific [17,22] or intraspecific variation [23] in orchids have also not found differentiation between populations that are geographically separated when analyzing floral characters with PCA.Such results are common given the method's assumptions, particularly the lack of a priori categorization into groups that would typically minimize intragroup variance and maximize intergroup variance [38].However, this multivariate method has been useful for recognizing morphotypes in orchid species represented by wild specimens [20] or cultivated ones [19,25] in Mexico.
The analysis of P. karwinskii's floral morphology with the CVA and cluster analysis were informative for discriminating infraspecific variation.These analyses showed that individuals from Alabarradas are well separated from the rest of the localities.Differences in the teeth of the column discriminate these individuals from those from other localities.Additionally, individuals from Jaltianguis and Yahuiche showed low dispersion and tended to overlap with each other.This morphological pattern is congruent with geography since these two locations are very close to each other in the region known as Sierra Norte of Oaxaca.Interestingly, individuals from Etla, the locality closest to the previous two, tend to show low dispersion among themselves and separate from those of Jaltianguis-Yahuiche.The morphological differentiation that is present with respect to Teposcolula is more expected.The CVA showed that the variables with the greatest contribution are traits of the column (height of the middle and the lateral teeth, and thickness at the level of the anther), lateral sepal (maximum width, total length), labellum (maximum width of the lateral lobe, total length, angle between the lateral lobe and claw, and maximum width).Due to the nature of the characters revealed by the analyses as the most important for the morphological patterns of the species, these traits could be related to the attraction of pollinators, as has been hypothesized in other studies analyzing morphological variation in other plants [22,39].We recommend paying attention to the most important variables presented in this work, as they can be useful as taxonomic markers at the intraspecific level and possibly at the level of the Prosthechea citrina complex, the species group to which P. karwinskii belongs.
The population of Albarradas turned out to be the most differentiated among those of P. karwinskii from Oaxaca.The individuals from this locality exhibit the lowest height of the middle and lateral teeth of the column, the smaller angle between the lateral lobe and claw of the labellum and the thinnest thickness of the column at the level of the anther.Additionally, they have the greatest maximum width of the lateral lobe of the labellum.This population could be recognized as a variety or geographic form of the species, geographically isolated in the central part of the state of Oaxaca.Moreover, the locality of Albarradas is a priority for the conservation of the species, as it represents a unique morphological variant.Unfortunately, the forest where this form grows in Albarradas hosts one of the least numerous populations of this orchid in Oaxaca.It will be interesting to verify if the morphological differentiation of the Albarradas population is related to genetic differentiation, as has been corroborated for other orchid species using vegetative characters [17].

Morphological Patterns of Zaachila Flowers of Unknown Origin
Among the natural populations of P. karwinskii used as reference in this study to compare flowers obtained from Easter celebrations in Zaachila (2017-2019), morphometric analyses revealed greater overlap and morphological similarity between the Zaachila material and populations from Yanhuitlan and Etla.Flowers from Zaachila are distinct from those in Albarradas, Jaltianguis and Yahuiche (Sierra Norte), Teposcolula (Mixteca), Juquila and Sola_El Lazo (Southern mountains of Oaxaca).Conversely, Zaachila specimens exhibit varying degrees of overlap with individuals from the Sierra Sur (Sola_Lachixío and Sola_Rancho Viejo) and Mixteca (Tlaxiaco) regions.While the species is relatively common across various forests in Oaxaca, our inference suggests that individuals rescued from Zaachila could come from the Mixteca or vicinity of Etla.However, the available material spans more than one year of festivities, indicating participants likely gathered flowers from multiple locations over three years.The findings presented are specific to the study period, and we caution that they may vary annually depending on where extraction occurs.
Morphometric methods have been successfully employed to determine the unknown origins of organisms in various animal groups, utilizing live specimens, preserved in museums or commercialized [26][27][28][29][30].However, they are relatively unexplored in plant species for this purpose.This study represents an initial attempt to infer the origins of Zaachila individuals based on morphological data.We point out the challenges (even with other types of data, e.g., genetics), including potential variance due to collection from multiple localities during the sampling period, gaps in samples from other distribution sites [26,28], and varying population sizes [40], which are intrinsic to orchids and other epiphytes [41].
Several populations studied here are situated in areas where specimens are harvested for local markets and religious use [3,8,9], impacting genetic variability and effective population sizes, as well increasing the chances of experiencing inbreeding depression and bottleneck events [13][14][15].Genetic analysis could help elucidate the causes of floral morphological variation in P. karwinskii and its biogeographical patterns, though correlations between genetic and morphological variability are not always straightforward.
Mexico's use of plants to satisfy aspects of the cultural and social life of local communities is vast, given the high cultural and biological diversity [42][43][44].Among them, ceremonial uses constitute a cultural element of the people, making their transmission important.However, the extraction of wild plants used in such ceremonies often has a negative impact on their populations [45,46].The conservation and use of wildlife are controversial [47], but these topics must be addressed through collective strategies [45,48].There are some preliminary initiatives for P. karwinskii [10,49].These efforts motivate us to develop collaborative conservation strategies that ensure the maintenance of the morphological and genetic variability of the species in regions most susceptible to flower extraction for traditional uses, as well as in the communities where these extracted specimens are destined.This will help guarantee the long-term preservation of both the species and its cultural significance.

Biological Material
During the flowering season of P. karwinskii (March-April), localities (populations) representing its distribution in Oaxaca were visited between 2015 and 2021.The geographical information of the sampled localities is presented in Table 3, while Figure 5 shows their geographical distribution on a map of Oaxaca.In each visited locality, one flower per individual was collected, ensuring they were on different host trees to avoid collecting ramets from the same individual; the proximal flower on the inflorescence was chosen when it had more than one.The sample size of each locality depended on the population abundance and is indicated in Table 3.A voucher specimen (herbarium or spirit) was prepared from each locality, which was deposited in the Herbarium OAX (acronym according to [50].Additionally, flowers rescued from specimens used as decorations in Catholic temples during the Easter celebration in the Villa de Zaachila, Oaxaca (2017-2019) were obtained.These samples, whose original locality is unknown, were obtained with permission from the organizing committee of this commemoration in the community in 2019, once they were removed from the temples.Both the flowers collected in the field and the rescued ones were preserved in a fixing solution of water (78%), ethanol 96% (21%), lactic acid 85% (6%), benzoic acid (0.5% w/v), and glycerin (5%), and then deposited in the Laboratory of Extraction and Analysis of Natural Products (CIIDIR Oaxaca, Instituto Politecnico Nacional).Table 3. Geographic information for the localities of Prosthechea karwinskii in Oaxaca, Mexico.See Section 4.2 to know how the expanded populations were integrated.QF = Quercus forest, QPF = Quercus-Pinus forest.n = number of individuals represented in the sample size per population.The locality/population numbers correspond to those shown in the map of Figure 5.The species is an orchid protected by Mexican environmental legislation; therefore, the coordinates of the localities were omitted.NA = the data does not apply.3 for additional information of the localities.

Selection of Floral Characters
Each flower of P. karwinskii was dissected into sepals, petals, labellum, an to select the characters used as variables in the morphometric analyses, accordin et al. [18] and da Cruz et al. [23] with some modifications.Photographs (Ca camera) were taken of the sepals, petals, and labellum, which were spread out in Figure 6A.The column was separated from the rest of the perianth for reco characters on this structure, as shown in Figure 6B.The ovary and column wer dinally sectioned to show the nectary or cuniculus and to record their characters in Figure 6C.Since the flower of P. karwinskii has bilateral symmetry, only the sepal, petal, lateral lobe of the labellum, and lateral tooth of the column were co A total of 45 characters were selected from these structures, of which 39 were lin urements and 6 were angles (Table 4, Figure 6).Each linear measurement was t a digital caliper, and the angles were measured with a protractor.Missing dat individuals for certain characters, due to herbivory, were replaced with the p average for that variable.The set of these continuous characters was recorded in 185 individuals of P. karwinskii.Specimens rescued from Zaachila were consid separate population.Specimens morphologically similar from populations ver  3 for additional information of the localities.

Selection of Floral Characters
Each flower of P. karwinskii was dissected into sepals, petals, labellum, and column to select the characters used as variables in the morphometric analyses, according to Borba et al. [18] and da Cruz et al. [23] with some modifications.Photographs (Canon Rebel camera) were taken of the sepals, petals, and labellum, which were spread out as shown in Figure 6A.The column was separated from the rest of the perianth for recording the characters on this structure, as shown in Figure 6B.The ovary and column were longitudinally sectioned to show the nectary or cuniculus and to record their characters, as shown in Figure 6C.Since the flower of P. karwinskii has bilateral symmetry, only the right-side sepal, petal, lateral lobe of the labellum, and lateral tooth of the column were considered.A total of 45 characters were selected from these structures, of which 39 were linear measurements and 6 were angles (Table 4, Figure 6).Each linear measurement was taken with a digital caliper, and the angles were measured with a protractor.Missing data in some individuals for certain characters, due to herbivory, were replaced with the population average for that variable.The set of these continuous characters was recorded in a total of 185 individuals of P. karwinskii.Specimens rescued from Zaachila were considered as a separate population.Specimens morphologically similar from populations very close to each other (<5 km apart) were integrated as a single population (expanded population); when a locality was represented by fewer than five specimens, they were also integrated with those from the nearest locality, thus avoiding the effect of a nonrepresentative population.Due to this, the CVA and cluster analysis included 12 expanded populations, as shown in Table 3.
x FOR PEER REVIEW 14 of 18  4 for the names of the variables.Photographs by R. Solano.

Statistial Analyses
An analysis of variance (ANOVA) and a Tukey test as a post-hoc analysis were conducted to find significant differences among each of the 45 floral characters and the origin locality of the individuals.Out of the 45 floral traits, 5 did not meet all the assumptions for applying an ANOVA, including normality.To assess if there are differences between populations for these five characters (angle at the apex of the middle lobe, thickness at the level of the anther, thickness in the middle part, width at the stigma level, and width of the middle tooth), a Kruskal-Wallis non-parametric test was applied, followed by Dunn's test with Bonferroni correction as a post-hoc analysis.These analyses were performed using R 2023.12.0 [51] in the stats package.
Multivariate analyses were implemented in Statistica 10 [52].First, a correlation test was conducted between all possible pairs of variables.Out of the 45 selected variables, 12 showed a correlation greater than 0.9 with each other and were therefore discarded for further analyses.By floral structure, the discarded variables were from the lateral sepal (width at 1/3 of its length, width at 2/3 of its length), dorsal sepal (total length, maximum  4 for the names of the variables.Photographs by R. Solano.

Table 4.
Floral morphologic variables of Prosthechea karwinskii and their coding used in the present study.The number and code of these variables correspond to those shown in Tables 1 and 2, as well as in Figure 6.

Statistial Analyses
An analysis of variance (ANOVA) and a Tukey test as a post-hoc analysis were conducted to find significant differences among each of the 45 floral characters and the origin locality of the individuals.Out of the 45 floral traits, 5 did not meet all the assumptions for applying an ANOVA, including normality.To assess if there are differences between populations for these five characters (angle at the apex of the middle lobe, thickness at the level of the anther, thickness in the middle part, width at the stigma level, and width of the middle tooth), a Kruskal-Wallis non-parametric test was applied, followed by Dunn's test with Bonferroni correction as a post-hoc analysis.These analyses were performed using R 2023.12.0 [51] in the stats package.
Multivariate analyses were implemented in Statistica 10 [52].First, a correlation test was conducted between all possible pairs of variables.Out of the 45 selected variables, 12 showed a correlation greater than 0.9 with each other and were therefore discarded for further analyses.By floral structure, the discarded variables were from the lateral sepal (width at 1/3 of its length, width at 2/3 of its length), dorsal sepal (total length, maximum width, width at 1/3 of its length, width at 2/3 of its length), petal (total length, width at 1/3 of its length, width at 2/3 of its length), labellum (width at 1/3 of its length, width of the base of the middle lobe), and column (thickness in the middle part).The remaining 33 characters were subjected to ordination analysis to evaluate morphological variation among populations, which allowed identifying the most significant variables for morphological patterns.To explore the data structure and detect possible outliers, a principal component analysis (PCA) based on the correlation matrix (correlated variables excluded) was performed.Since this exploratory analysis did not show the presence of outliers, no individuals were removed from the analyses.Subsequently, a canonical variate analysis (CVA) was performed using the expanded populations as categorical variables.We used the standardized coefficients for canonical variables to identify the most important in the observed patterns [18].Both the correlation test, PCA, and CVA were conducted including and excluding samples from Zaachila (of unknown origin).We calculated a matrix of squared Mahalanobis distances between the expanded population centroids (including Zaachila), using the expanded populations as the group variable and the 33 floral variables.This matrix was used to perform a cluster analysis using UPGMA (unweighted pair-group method) with arithmetic averages as the clustering algorithm.

Conclusions
Through morphometric analysis, we examined the intraspecific variation in the floral morphology of P. karwinskii, identifying characters as potential taxonomic markers for the species related to the P. citrina complex.Traits from lateral sepal, labellum, and column, are useful for this purpose and they are related to the morphological patterns of P. karwinskii.The majority of floral characters analyzed here varied significantly among populations of P. karwinskii.The CVA was informative for discriminating the infraspecific variation in this orchid, but the PCA was not.Albarradas harbors the most differentiated population of P. karwinskii in Oaxaca, and it might be recognized as a variety or geographical form of the species and thus considered a priority for conservation.The Teposcolula population has value for horticultural management due to its individuals having the largest flowers in this species.
The results show that the floral characters have the potential to associate specimens of unknown origin with their probable geographical region.The individuals rescued in the Zaachila group with those from Etla and Yanhuitlan suggest that the origin of the former might be assigned to these two localities or others very close to them.Additional studies are recommended to compare morphological variation with genetic and geographical variation of the populations studied here.Finally, this study constitutes an initial attempt to determine the unknown geographical origin of an orchid extracted for religious use by Mexican communities.The method employed here is low-cost, allows the analysis of a large number of individuals, and could be applied to other orchid species.However, the results must be interpreted with caution, as the accuracy of traceability depends on a good reference and a careful analysis.

Figure 1 .
Figure 1.(A) Habitat of Prosthechea karwinskii, an oak forest in San Pedro y San Pablo Teposcolula, Oaxaca.(B) Prosthechea karwinskii growing in situ as a hanging epiphyte on Quercus sp. in Santo Domingo Yanhuitlan, Oaxaca.Photographs by R. Solano.

Figure 1 .
Figure 1.(A) Habitat of Prosthechea karwinskii, an oak forest in San Pedro y San Pablo Teposcolula, Oaxaca.(B) Prosthechea karwinskii growing in situ as a hanging epiphyte on Quercus sp. in Santo Domingo Yanhuitlan, Oaxaca.Photographs by R. Solano.

Figure 2 .
Figure 2. Representation of axes 1 and 2 of the PCA resulting from the variation of 33 floral varia in 185 individuals of Prosthechea karwinskii from 18 localities (including Zaachila) in Oaxaca, Me

Figure 2 .
Figure 2. Representation of axes 1 and 2 of the PCA resulting from the variation of 33 floral variables in 185 individuals of Prosthechea karwinskii from 18 localities (including Zaachila) in Oaxaca, Mexico.

Figure 3 .
Figure 3. Representation of the axes 1-2 (A) and 1-3 (B) of the CVA resulting from the variation of 33 floral variables in 185 individuals of Prosthechea karwinskii from 12 expanded populations in Oaxaca, Mexico.See Section 4 to see how the expanded populations were integrated.

Figure 3 .
Figure 3. Representation of the axes 1-2 (A) and 1-3 (B) of the CVA resulting from the variation of 33 floral variables in 185 individuals of Prosthechea karwinskii from 12 expanded populations in Oaxaca, Mexico.See Section 4 to see how the expanded populations were integrated.

Figure 4 .
Figure 4. Phenogram showing the relationships between 12 expanded populations of Prosthechea karwinskii from Oaxaca, Mexico.The dendrogram was constructed using the UPGMA clustering algorithm and the squared Mahalanobis distances between the population centroids, calculated from 33 floral variables.

Figure 4 .
Figure 4. Phenogram showing the relationships between 12 expanded populations of Prosthechea karwinskii from Oaxaca, Mexico.The dendrogram was constructed using the UPGMA clustering algorithm and the squared Mahalanobis distances between the population centroids, calculated from 33 floral variables.

Figure 5 .
Figure 5. Localities where individuals of Prosthechea karwinskii were sampled in Oaxaca, M Table3for additional information of the localities.

Figure 5 .
Figure 5. Localities where individuals of Prosthechea karwinskii were sampled in Oaxaca, Mexico.See Table3for additional information of the localities.

Figure 6 .
Figure 6.Floral structures showing the variables evaluated in the morphometric analyses of the 18 populations (including Zaachila) of Prosthechea karwinskii from Oaxaca, Mexico.(A) Flower dissection.(B) Lateral and ventral views of the column (C) Longitudinal section of the column and ovary, showing the cuniculus.Sl = lateral sepal, Sd = dorsal sepal, Pe = petal, La = labellum, Co = column, Dl = lateral tooth of the column, Dm = median tooth of the column, Cu = cuniculus.See Table4for the names of the variables.Photographs by R. Solano.

Figure 6 .
Figure 6.Floral structures showing the variables evaluated in the morphometric analyses of the 18 populations (including Zaachila) of Prosthechea karwinskii from Oaxaca, Mexico.(A) Flower dissection.(B) Lateral and ventral views of the column (C) Longitudinal section of the column and ovary, showing the cuniculus.Sl = lateral sepal, Sd = dorsal sepal, Pe = petal, La = labellum, Co = column, Dl = lateral tooth of the column, Dm = median tooth of the column, Cu = cuniculus.See Table4for the names of the variables.Photographs by R. Solano.

Table 1 .
Results of the analysis of variance (ANOVA) for 40 of the 45 floral characters that met the assumption of normality recorded in 185 individuals from 12 expanded populations of Prosthechea karwinskii, including Zaachila.df = degrees of freedom, ss = sum of squares, ms = mean square.Differences are significant at p ≤ 0.05, number of asterisks indicates increasing levels of significance, a dot indicates that the test value was not significant.

Table 2 .
Contribution between variables and axes with eigenvalues > 1.00 of PCA (18 populations, including Zaachila) and CVA (12 expanded populations) of Prosthechea karwinskii from Oaxaca, Mexico.Values in bold correspond to the three highest for each axis (A).